Chemical analysis method for multiplexed samples

ABSTRACT

Analyzing a plurality of fluid specimens with a single analyzing instrument comprising introducing different combinations of specimens into a homogenizing volume to create a homogenized specimen and with a programmed digital computer mathematically processing the recorded results to produce analyses corresponding to individual fluid specimens.

FIELD OF THE INVENTION

This invention relates to analysis of large numbers of fluid specimensin instruments, such as mass spectrometers with a single instrument.

BACKGROUND OF THE INVENTION

One prior method of analyzing large numbers of specimens with a singleinstrument is with a multiplexed sampling system wherein samples areprepared and supplied to the instrument one at a time. For example,several electrospray needles and a rotating barrier with a hole thatallows sprayed fluid streams emerging from the needles to be sampled oneat a time has been proposed. Another approach is to provide a gassampler that sequentially diverts one of a plurality of gas streams toan instrument. One commercially available selector valve performs rapidsample switching between up to 40 sample streams. The problem witheither of these multiplexed sampling approaches is that only onespecimen is analyzed at a time. To improve the signal-to-noise ratio ofthe results of the analysis, it is necessary to repeat each sample overand over again.

According to this invention, Hadamard transform or another transformtechnique is used to analyze multiple specimens simultaneously. Thisimproves the signal-to-noise ratio by a factor of:$\frac{\left( {N + 1} \right)/2}{N^{1/2}}$for N separate specimens over the same measurement time or it wouldreduce the time 4/N to obtain the same signal-to-noise ratio as theindividual measurement approach.

The Hadamard transform method is well known in spectroscopy and it isessentially based on solving n simultaneous equations in n unknowns todeconvolute the stored results. Hadamard transform methods have beenused in MS/MS experiments in a Fourier transform mass spectrometer asexplained by Loh, Williams, McLafferty and Cody in “Simultaneous MS-IIMeasurements Using Hadamard Transform Fourier Transform MassSpectrometry”, Analytical Chemistry (1988). In that case, differentcombinations of precursor ions were selected for dissociation. From theresulting spectra, individual daughter spectra were obtained by solvingsimultaneous equations. The Hadamard transform method has also beenapplied to time-of-flight mass analyzers wherein multiple testingconditions are simultaneously used with the same specimen followed bydeconvolution with Hadamard transforms as set forth in Franzen U.S. Pat.No. 5,719,392.

SUMMARY OF THE INVENTION

It is an advantage, according to this invention, to provide amultiplexed sampling method wherein a plurality of fluid samples areanalyzed simultaneously to improve the signal-to-noise ratio for a giventime period or to shorten the time period for a given signal-to-noiseratio.

Briefly, according to this invention, there is provided a method foranalyzing a plurality of fluid specimens with a single analyzinginstrument. It comprises the steps for:

-   -   a) preparing a plurality of N fluid specimens,    -   b) introducing a first combination of r specimens wherein r is        less than N into a homogenizing volume to create a homogenized        specimen,    -   c) introducing at least a portion of the homogenized specimen to        the analyzing instrument and recording the results of the        analysis maintaining an association with the combination of r        specimens,    -   d) introducing additional different combinations of specimens        into said homogenizing volume and repeating steps b) and c), and    -   e) with a programmed digital computer mathematically processing        the recorded results to produce analyses corresponding to        individual fluid specimens.

In one embodiment, the fluid specimens are gaseous specimens dilutedwith a carrier gas and the analyzing instrument is a mass spectrometer.The mathematical processing comprises deconvolution wherein themathematical processing comprises a Hadamard transform.

According to a preferred method, each specimen is directed into thehomogenizing volume from individual nozzles connected to electronicallycontrolled valves. The nozzle sizes, pressure drops therethrough, andopen times of the valves are controlled to introduce a specified mass ofeach fluid specimen into the homogenizing volume. Normally, when nozzlesare not supplying fluid specimen to the homogenizing volume, the flow ofthe specimen is diverted and continued.

Preferably, the number of specimens N is an odd number greater than 2and r is an even number equal to (N+1)/2.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and other objects and advantages will become clear fromthe following detailed description made with reference to the drawingsin which:

FIGS. 1(a), 1(b), and 1(c) illustrate the use of a rotating mask toselect groups of fluid samples; and

FIG. 2 is a section view through a rotating selector for selectinggroups of gas samples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention has application to mass spectrometry, for example. A massspectrometer produces ions from chemical substances that are to beanalyzed. The mass spectrometer then uses electric and magnetic fieldsto measure the mass of the charged particles. The masses and therelative abundance of the ions in a mass spectrum can be used todetermine the structure and composition of molecules. A magnetic sectoranalyzer (just one form of mass spectrometer) separates ions accordingto their momentum (the product of their mass times their velocity). Anelectric sector analyzer separates the ions according to their kineticenergy. Both magnetic sectors and electric sectors are used in the highresolution double-focusing mass spectrometers. In its simplest mode ofoperation of the double-focusing mass spectrometer, the ions areaccelerated at a constant potential into the electric sector, theelectric sector is maintained at a constant potential, and the strengthof the magnetic sector is varied. As the field strength of the magneticsector is swept, ions of different mass-to-charge ratios are brought tofocus on a detector slit. The detector counts the ions passing throughthe slit and the count versus the field strength (which in turncorrelates to mass-to-charge ratio) comprises the mass spectrum. In thesimple case, ions from only one sample at a time are accelerated intothe electric field. Mass spectra can be gathered using other types ofmass spectrometers, for example, quadrupole mass spectrometers,time-of-flight mass spectrometers, quadrupole ion trap massspectrometers, and Fourier transform mass spectrometers. As with thedouble-focusing mass spectrometers, one sample at a time is tested.

As already explained, a chemical compound or fragment thereof must beionized in order to be analyzed by mass spectrometry. Any number ofionization methods are used, for example, electron impact ionization,chemical ionization, field ionization, and fast atom bombardment, tomention just a few. In each case, the sample is passed into anionization chamber and ions are drawn out of the chamber and acceleratedinto the mass spectrometer. According to this invention, more than onesample at a time is introduced into the ionization chamber. It is notnecessary that each specimen have identical mass as each other specimen,but it is necessary that each time a specimen is introduced, the samemass is introduced. The combinations of samples to be passed into theionization chamber are selected according to Hadamard techniques. Thesimplest case would be introduction of three samples, two at a time. Inthis case, three different mass spectrums would be gathered, none ofwhich would be the spectrum of any one of the samples. The threespectrums are digitized and stored in a computer database. They can thenbe deconvoluted by mathematical techniques.

Several techniques are possible for physically combining fluid, and moreparticularly, gas samples prior to introduction into the ionizationchamber. One implementation comprises using a plurality of electrosprayneedles and a rotating barrier with a mask having openings that pass aselected number of sprays at any given time to the center thereof wherethey can be mixed and channeled to the ionization chamber. FIGS. 1(a),1(b), and 1(c) schematically illustrate the rotating mask at threepositions for the trivial case of three spays, one for each of threesamples.

FIG. 2. schematically shows a diversion valve for selecting samples.This is a prior art valve that was originally designed to pass onesample at a time modified to pass multiple samples at one time. Therotor is provided with multiple sample inlet connections instead of onlya single sample inlet connection. Referring to FIG. 2, a rotor 10 isdriven by shaft 11 and drive coupling 12, and drive motor and encoder13. The rotor is provided with a plurality of sample transfer passages15 for diverting sample flow from sample inlets to the sampling probe16. The non-selected sample flow exhausts to an exhaust annulus 17 thatdelivers the mixed non-selected samples to a waste exhaust 18.

Applied Bioanalytical has demonstrated a microchip device having spraysthat can be switched on or off electronically, so a “mask” could beomitted and the spray combinations could be generated electronically.

Let a, b, and c represent the results of measuring spray channels A, B,and C independently, and let x, y, and z represent the results of thecombined sprays in steps 1, 2, and 3, respectively in the three sampleexample. If we represent the example above in matrix notation,$\left( \quad\begin{matrix}x \\y \\z\end{matrix}\quad \right) = {\left( \quad\begin{matrix}1 & 1 & 0 \\1 & 0 & 1 \\0 & 1 & 1\end{matrix}\quad \right)\left( \quad\begin{matrix}a \\b \\c\end{matrix}\quad \right)}$

The original results can be obtained by using an inverse matrix:$\left( \quad\begin{matrix}a \\b \\c\end{matrix}\quad \right) = {\left( \quad\begin{matrix}1 & 1 & {- 1} \\1 & {- 1} & 1 \\{- 1} & 1 & 1\end{matrix}\quad \right)\left( \quad\begin{matrix}x \\y \\z\end{matrix}\quad \right)}$

The improvement in signal-to-noise in the three sprayer case is:$\frac{2}{\sqrt{3}} = 1.15$

The improvement is greater for larger numbers of spray nozzles. Forseven sprays, the improvement in signal-to-noise is:$\frac{4}{\sqrt{7}} = 1.51$for the sample measurement time as the individual measurements, or thesame signal-to-noise ratio could be obtained in roughly half ({fraction(4/7)}) the time.

Even numbers of sprays are not suitable for this method, so a 96-spraydevice would have to be modified to a 95-spray device. For a 95-spraydevice, the improvement in signal-to-noise would be:$\frac{48}{\sqrt{95}} = 4.9$or the same signal-to-noise ratio could be obtained in {fraction(4/95)}=0.04 the time required for individual measurements.

One can imagine extending this concept to other multiple samplingapplications. One such example is matrix-assisted laser desorptionionization (MALDI). In MALDI, multiple samples are placed on plates with(for example) 96 sample spots per plate. Samples are typically measuredone at a time by firing a laser at the spot and using a time-of-flightmass spectrometer to analyze the ions produced by laser desorption. Itis common practice to average multiple laser shots per spot to get goodsignal-to-noise ratios. One can imagine firing multiple laser beams (ora split laser beam) at the sample spots in combinations defined byHadamard transform principles and then solving for the spectra from eachindividual spot with the resulting gain in signal-to-noise ratios, or areduction in analysis time.

The method described herein is applicable to other analytical methodswherein multiple fluid streams can be sampled and combined for analysis.

1. A method for analyzing a plurality of fluid specimens consisting essentially of compounds and fragments thereof with a single analyzing instrument wherein multiple fluid streams can be sampled and combined comprising the steps for: a) preparing a plurality of N fluid specimens wherein N is an odd number greater than 2; b) introducing a first combination of r specimens wherein r is an even number equal to (N+1)/2 into a homogenizing volume to create a homogenized specimen; c) introducing at least a portion of the homogenized specimen to the analyzing instrument and recording the results of the analysis maintaining an association with the combination of r specimens; d) introducing additional different combinations of specimens into said homogenizing volume and repeating steps b) and c); and e) with a programmed digital computer mathematically processing the recorded results to produce analyses corresponding to individual fluid specimens.
 2. The method according to claim 1, wherein the fluid specimens are gaseous specimens diluted with a carrier gas.
 3. The method according to claim 2, wherein the analyzing instrument is a mass spectrometer.
 4. The method according to claim 3, wherein the mathematical processing comprises deconvolution.
 5. The method according to claim 4, wherein the mathematical processing comprises a Hadamard transform.
 6. The method according to claim 1, wherein each specimen is directed into the homogenizing volume from individual nozzles connected to electronically controlled valves.
 7. The method according to claim 6, wherein the nozzle sizes, pressure drops therethrough, and open times of said valves is controlled to introduce a specified mass of each fluid specimen into the homogenizing volume.
 8. The method according to claim 7, wherein when the nozzles are not supplying specimen to the homogenizing volume the flow of the specimen is diverted and continued. 